Multi-class supercritical fluid extraction of bioactive compounds from Citrus leaves: Development of a unified strategy for phenols, carotenoids, oxygen heterocyclic compounds and tocopherols

A supercritical fluid extraction strategy was developed and optimized for the simultaneous recovery of multiple classes of bioactive compounds from Citrus leaves, including phenols, carotenoids, oxygen heterocyclic compounds, and vitamin E. Selective ultrasound-assisted extraction methods were first employed, highlighting limitations related to different protocols, the use of toxic solvents, and limited process integration. To overcome these drawbacks, supercritical fluid extraction conditions were systematically optimized by modulating co-solvent composition, temperature, and extraction time through a multi-step approach. The optimized method, based on sequential dynamic extraction with 10% bioethanol followed by 30% bioethanol/water (80:20, v/v) at 350 bar and 70 °C, enabled efficient extraction across a wide polarity range, achieving recovery values of 91, 101, 92 and 82% for phenols, carotenoids, oxygen heterocyclic compounds and vitamin E, respectively, in lemon leaves. The method was successfully applied to orange and mandarin leaves, providing comparable quantitative profiles without the need for matrix-specific optimization. Chromatographic analyses confirmed Citrus leaves as a valuable source of bioactive compounds, with phenols as the predominant class. Overall, the proposed supercritical fluid extraction approach represents a simple, flexible, and sustainable alternative to conventional extraction techniques for comprehensive metabolite profiling.